Canadian Journal of Forest Research

Desiccation, dormancy and storage of fraxinifolia () seeds: application in Hyrcanian and Colchis Forest conservation

Journal: Canadian Journal of Forest Research

Manuscript ID cjfr-2018-0519.R2

Manuscript Type: Article

Date Submitted by the 09-Sep-2019 Author:

Complete List of Authors: Wawrzyniak, Mikolaj; Polska Akademia Nauk Instytut Dendrologii w Korniku, Reproductive Biology and Population Genetics Jasińska, Anna; Polska Akademia Nauk Instytut Dendrologii w Korniku, LaboratoryDraft of Systematics and Geography Chmielarz, Pawel; Polish Academy of Sciences Kozlowski, Gregor; Universite de Fribourg, Department of Biology; Museum Fribourg, Natural History

biodiversity, Georgia, critical moisture content, cryopreservation, seed Keyword: banking

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

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1 Desiccation, dormancy and storage of (Juglandaceae) seeds:

2 application in Hyrcanian and Colchis Forest conservation

3 Mikołaj Wawrzyniak1,*, Anna Katarzyna Jasińska2, Paweł Chmielarz1, Gregor Kozlowski3,4

4 1Department of Reproduction Biology and Population Genetics, Institute of Dendrology, Polish

5 Academy of Sciences, Parkowa 5, 62-035, Kórnik, Poland

6 2Laboratory of Systematics and Geography, Institute of Dendrology, Polish Academy of

7 Sciences, Parkowa 5, 62-035, Kórnik, Poland

8 3Department of Biology, University of Fribourg, Chemin du Musée 10, CH-1700 Fribourg,

9 Switzerland

10 4Natural History Museum Fribourg, Chemin du Musée 6, CH-1700 Fribourg, Switzerland 11 *Corresponding author. E-mail address:Draft [email protected] (M. Wawrzyniak) Tel.: 12 +48 61 81 70 033; ORCID: https://orcid.org/0000-0002-4297-5741

13

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14 Abstract

15 Pterocarya fraxinifolia (Juglandaceae) is a model relict tree species native to South Caucasus

16 and it is a typical element of threatened riparian forests. Intensive land transformations which

17 is common in Transcaucasia, resulted in loss of natural habitat and population decline of the

18 species. One of the method for ex situ conservation is seed banking. Cryopreservation in liquid

19 nitrogen (LN; -196°C) is particularly of interest as it allows safe preservation of valuable

20 genetic resources. However, the feasibility of seed cryopreservation is related to their

21 desiccation tolerance and intrinsic composition. In this study, we examined the physiological

22 P. fraxinifolia seed traits from which desiccation tolerance is unknown or controversial and

23 their feasibility to cryopreservation,. Additionally, we tested stratification methods for 24 dormancy assessment. Results showedDraft that seeds survived desiccation to a 2.8% seed moisture 25 content (MC) and germinated in 64% . Stratification at temperature 3°C for 8 weeks was proven

26 to be both fast and effective. For cryopreservation, seed MC ranging between 2.8% and 18.1%

27 was determined to be safe. There was no difference in seedling emergence in seeds stored for

28 one year regardless of the storage temperature (-3°, -18° or -196°C). Based on our results, P.

29 fraxinifolia seeds can be classified as orthodox. This study demonstrates for the first time the

30 feasibility of cryopreserving P. fraxinifolia seeds.

31 Keywords: seed banking, cryopreservation, biodiversity, critical moisture content, Georgia

32

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33 Introduction

34 The conservation of plant genetic resources is an established and globally recognized priority

35 because the variability of species is fundamental to forestalling their extinction. It is estimated

36 that there are more than 300,000 species of land , all of which are essential for the proper

37 functioning of local ecosystems (Corlett 2016). However, many populations of plants are

38 currently threatened by loss, fragmentation and degradation of habitats as well as

39 overexploitation, invasive species and climate change (Groom et al. 2005). Conservation

40 programs for threatened species can follow a variety of criteria, i.e., economic value, ecological

41 value or species uniqueness. One of the unique and irreplaceable parts of the plant kingdom is

42 relict trees. These surviving remnants have been present on the Earth for millions of years, 43 representing evolutionary processes andDraft outlasting environmental, biogeographical and climate 44 changes. Despite their continued existence over millions of years, they usually retain a narrow

45 distribution in so-called refugia (Maharramova et al. 2017; Kozlowski et al. 2018) such as

46 Hyrcanian area in the North Iran. The unique ecosystems of Hyrcanian Forests along with plains

47 around the Black Sea (Colchis Forests) are one of the most valuable biosphere genetic resources

48 in the World, which are a shelter of many relict species such as: Parrotia persica (DC.) C. A.

49 Mey., Gleditsia caspica Dest., Zelkova carpinifolia (Pall.) K. Koch. and Pterocarya fraxinifolia

50 (Poir.) Spach. (Naqinezhad et al. 2018).

51 The possibility of in situ conservation has its limitations. A holistic approach to

52 conservation is often difficult to fund and maintain (Diamond 1989), therefore, ex situ

53 conservation, such as seed banking, seem to be most suitable for seed bearing species, as it

54 provides inexpensive and effective long time strategy (Bewley et al. 2013; Zaritzky 2015). The

55 viability of stored seeds depends on the seed moisture content and storage temperature

56 (Vertucci and Roos 1990). Seed storability at low temperature depends on its desiccation

57 tolerance, as access water can crystallize and cause intracellular damage (Wesley-Smith et al.

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58 2014). The practical categorization proposed by Roberts (1973) and then supplemented by Ellis

59 et al. (1990) considers three categories of seeds divided on the basis of their desiccation

60 sensitivity: orthodox (seeds tolerate desiccation below a 5% moisture content, MC, fresh-

61 weight basis), recalcitrant (seeds that do not tolerate desiccation), and intermediate (seeds that

62 exhibits characteristic of both former categories i.e. seeds that tolerate moderate desiccation but

63 lose viability during subsequent storage at subzero temperatures). However, careful study of

64 seeds physiology is important; seeds storage behavior is often more complex as cellular

65 response on water and temperature thresholds create more in-between responses (Walters 2015).

66 Cryopreservation in liquid nitrogen (LN, between -130 and -196 °C) represents a safe

67 and cost-effective option for the long-term storage of germplasm. In recent years,

68 cryopreservation has become a successful method for conserving unique (i.e., endemic,

69 endangered, and economically valuable)Draft species, both as vegetative tissues such as dormant

70 buds and shoot tips and generative organs such as seeds and embryos (Engelmann 2011;

71 Lambardi and Shaarawi 2017). For orthodox seed species, cryopreservation is an alternative to

72 traditional storage at -20 °C and is of particular importance for long-term storage (10 to 100s

73 of years; Reed 2008), and it is the only method for the long-term storage of intermediate and

74 recalcitrant seed species. Cryopreservation of the seeds of woody species has proved to be

75 effective in both orthodox and recalcitrant seeds in many tree species, such as Alnus glutinosa,

76 (Chmielarz , 2010a, ), Coffea spp. (Dussert et al. 1998), Prunus armeniaca (Malik and

77 Chaudhury 2010), Prunus padus (Popova et al. 2016) and others. Together with the ex situ

78 approach, cryopreservation has been successfully utilized in conservation programs for

79 herbaceous plants, such as Cosmos atrosanguinea (Wilkinson 2002) or Lomandra sonderi

80 (Menon et al. 2014). Cryopreservation together with modern biotechnology has been

81 implemented in the conservation programs of many national floras, such as those of Brazil

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82 (Pilatti et al. 2011), Spain (González-Benito and Martín 2011), Australia (Ashmore et al. 2011)

83 and others.

84 In our study, we examined Caucasian wingnut (Pterocarya fraxinifolia (Poir.) Spach.)

85 of the walnut family (Juglandaceae) as a model relict tree species which belongs to the so-called

86 Arcto-Tertiary relict trees and grows in the lowlands and riparian habitats of the Southern

87 Caucasus, Anatolia and Caspian (Hyrcanian) forests of Iran, Georgia, Turkey and Azerbaijan

88 (Boratyński and Boratyńska 1975; Kozlowski et al. 2018). During the Oligocene and Miocene,

89 the species was widespread in the Northern Hemisphere, but due to climate cooling over the

90 last 15 million years, it became restricted to southern refugial areas characterized by a more

91 stable climate (Manchester 1987; Milne and Abbott 2002; Maharramova et al. 2017). In some

92 locations, the population growth of the species depends on individual clonal reproduction via

93 root sprouts, as natural regeneration viaDraft seeds is poor, and a majority of the seeds is empty (up

94 to 90%; Yilmaz 2015). Its populations are characterized by intermediate to low genetic diversity

95 (Akhani and Salimian 2003; Yilmaz 2015; Maharramova et al. 2017; Yousefzadeh at al. 2018)

96 and there is a danger that if the habitats of this species continue to degrade, it may, similar as

97 in Alnus and other riparian species (Barsoum et al. 2004; Dering et al. 2017), lead to a

98 significant increase in clonality. Although some of the populations are protected within national

99 parks in Georgia, Azerbaijan, and Iran, more protection is necessary—especially considering

100 edge and standalone populations—to preserve the genetic diversity of the Caucasian wingnut

101 (Kozlowski et al. 2018). In the scientific literature, there are a lack of comprehensive studies

102 on the seed traits (storage behavior, desiccation tolerance, dormancy) of Caucasian wingnut

103 regarding the preservation of genetic diversity. Seeds of Caucasian wingnut have physiological

104 dormancy and its release require at least 5 weeks of cold stratification in 4°C without medium

105 (Cicek and Tilki 2008), as control seeds did not germinate without stratification. However, no

106 detailed studies on dormancy release for seeds from natural stands were conducted as well

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107 information about dormancy in other Pterocarya species is sparse and indeterminate.

108 According to the Seed Information Database (Royal Botanic Gardens Kew 2018), seeds of the

109 Caucasian wingnut are considered to be the orthodox type despite the lack of information on

110 their ability to tolerate desiccation below 5% MC and storage viability in sub-zero temperatures.

111 In general knowledge of storage behavior in Juglandaceae family is understudied and

112 sometimes outdated. It is crucial to understand seed traits of those species to develop and

113 implement long-term methods of conservation i.e. cryopreservation. However, seed tolerance

114 to storage at low (sub-zero) temperatures (-3°, -18° and -196°C) is unknown for Pterocarya

115 fraxinifolia. The aim of the present study was to examine the biological traits of Caucasian

116 wingnut seeds, such as their desiccation tolerance and dormancy type and tolerance to exposure

117 to sub-zero temperatures, to establish an effective method for the conservation of its genetic

118 resources. More specifically, we examinedDraft (1) the critical moisture content (CMC) of Caucasian

119 wingnut seeds, (2) the high moisture freezing limit (HMFL) for cryopreserved seeds, (3) the

120 stratification methods (with and without a medium) and (4) the viability of the seeds after one

121 year of storage at three different temperatures (-3, -18 and -196 °C).

122 Materials and methods

123 Collection and moisture content

124 Mature fruits of P. fraxinifolia were collected in September 2016 from 20 individual trees

125 growing along Ninoskhevi River (minimum 20 meters from each other) in Ninigori, Eastern

126 Georgia (Southern Caucasus, 41°49'52"N, 46°12'28"E) and transferred to the lab. The

127 semiorbicular wings were wiped off with sand during cleaning, and all remaining impurities

128 were removed using sieves. Seeds were collected dry and had initial moisture content (MC)

129 11.5%. The term “nutlet” used in this paper refers to the true fruit of P. fraxinifolia (the central

130 part of the fruit, with a hard pericarp that is enclosed in the fleshy and green tissue that is issued

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131 from the fused floral elements, mainly the bracteoles (Fig. 1A, B; Schaarschmidt 2006).The

132 MC was determined separately for (1) the nutlet, containing the seed and pericarp, and (2) the

133 seed only. However, for practical reasons, the seed MC used throughout this paper applies to

134 the nutlets. The MC was calculated on a fresh-weight basis (%) using the formula:

(퐹푊1 ― 퐷푊)100 135 MC1 = 퐹푊1

136 where MC1 is the moisture content, FW1 is the initial fresh weight and DW is the dry weight

137 after drying.

138 The MC of the nutlets used for cryopreservation was adapted (either by drying or

139 moisturizing) to obtain 10 levels of MC ranging from 3 to 30% with increments of 140 approximately 3%. Nutlets with MC >10%Draft were moisturized several times with water to obtain 141 a higher MC, up to 30%. A lower MC was obtained after desiccation over activated silica gel.

142 Adjusting the MC of the nutlets was based on the FW of the moisturized or desiccated seeds

143 according to the formula:

1 퐹푊 (100 ― 푀퐶1) 144 퐹푊2 = 100 ― 푀퐶2

145 where FW2 is the desired weight and MC2 is the desired moisture content. After obtaining the

146 desired MC, the nutlets were left in tightly closed containers for 3-5 days at 3 °C to even out

147 the MC.

148 After reaching the weight of the desired MC level, the exact MC was determined by

149 drying the nutlets at 103 ± 2 °C for 17 hours (3 replicates of 10 nutlets). Critical moisture

150 content of seeds (CMC) was determined by germination test of the lowest level of tested MC

151 (2.3%). High moisture freezing limit (HMFL) was set for the highest seed MC tolerating

152 cryopreservation.

153 Stratification and germination test

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154 To break the dormancy of the seeds, the nutlets of Caucasian wingnut underwent cold

155 stratification at 3 °C after storage. In our experiment, we used four different stratification

156 methods to determine the most sufficient method that could be used later in a cryopreservation

157 experiment: (A) 12 weeks at 3 °C in a stratification medium; (B) 8 weeks at 3 °C in a

158 stratification medium; (C) 12 weeks at 3 °C without a stratification medium, soaking the seeds

159 for one hour every week and, after 4 weeks of stratification, soaking the seeds every two weeks;

160 and (D) 12 weeks at 3 °C without a stratification medium after moisturizing the nutlets up to

161 40% MC; and (E) submergence in LN for 48 hours and stratification for 12 weeks at 3 °C.

162 The stratification medium used in our experiment was a moist mixture (v/v, 1:1) of

163 sieved peat (pH 3.5-4.5) and quartz sand fraction (< 1 mm). Stratification without a substrate

164 occurred in plastic boxes with a lid. The seeds in method C were soaked in tap water for one

165 hour. Then, the water was drained fromDraft the boxes while the moist seeds were covered with a

166 lid and kept inside the box. Subsequently, the box with wet seeds was placed at 3 °C. The

167 moisture of the stratification medium and the visual condition of the nutlets were checked every

168 week regardless of the stratification method used.

169 The stratification medium was mixed with the nutlets in a 3:1 ratio and placed in 25 ml

170 plastic bottles with a lid. Each lid had three 0.5 cm diameter holes allowing gas exchange. The

171 condition of the nutlets and the substrate was monitored on a weekly basis to avoid soil drying.

172 The seed germination test for all experiments was performed after stratification in the same soil

173 substrate as described earlier. The germination test was conducted in darkness and cyclically

174 alternating the temperature between 3 and 20 °C (16 and 8 hours a day, respectively). Each

175 week, the germinated seeds were counted and discarded. After 10 weeks, the remaining nutlets

176 were cut to examine seed viability.

177 A seedling emergence test was also conducted in the same soil mixture of peat and sand

178 that was used in the germination test. After stratification, the nutlets were sown in plastic boxes

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179 into the substrate at a depth of 1 cm and covered with a layer of sand. To avoid the severe drying

180 of the substrate, the boxes were covered with a transparent plastic lid. The seedling emergence

181 test was conducted under the same thermal conditions as the germination test (3 and 20 °C, 16

182 and 8 hours a day) until the seedling reached ca. 2 cm in height. Then, the boxes with the

183 seedlings were moved into the light (60 µmol m-2 s-1 for 8 hours a day) in a chamber with a

184 controlled temperature of 20 °C. The experiment investigating the seed cryopreservation and

185 storage time consisted of 3 replications per 30 nutlets, and that evaluating the best stratification

186 method consisted of 4 replications per 50 nutlets.

187 Cryopreservation and storage

188 Before freezing (at -196 °C), the nutlets were placed in a plastic bag and tightly sealed. The

189 material was frozen by direct immersion in liquid nitrogen (LN) for 48 hours. Subsequently,

190 plastic bags with the material were thawedDraft at 42 °C in a water bath for 5 min.

191 The nutlets were stored at temperatures of -3 and -18 °C and in LN for two weeks and

192 for one year in 11.8% of MC. The nutlets that were not stored were used as a control treatment.

193 Statistical analyses

194 Data were analyzed using R statistical computing software (R Core Team 2017). We used

195 analysis of variance (ANOVA) to analyze significant differences between the mean values and

196 Tukey’s test for the pairwise comparisons. Mean value from each variant were calculated from

197 three replicates consisted from seeds sample. Each replicate was placed in separate box,

198 randomly placed in incubator/dewar (one incubator per each temperature). Analyses were

199 performed after Bliss transformation (arc-sine) of the proportional data. As fixed effects in one-

200 way ANOVA we analyzed seed treatment used in each experiment. For cryopreservation

201 experiment we used two-way ANOVA with integrations for moisture content level and liquid

202 nitrogen treatment . ANOVAs and Tukey’s tests were performed separately for the germination

203 and emergence tests.

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204 Results

205 Seeds moisture content, stratification and germination

206 The highest germination (70.5%) was obtained after submerging the seeds in liquid nitrogen

207 (LN) for 48 hours prior to 12 weeks of cold stratification (Fig. 2). Stratification for 12 weeks at

208 3 °C without a stratification medium and periodic submersion in water for moisturization

209 resulted in a significantly lower germination of 40.5% and the highest number of decayed seeds

210 (37%; Table 1). The other tested methods of stratification (A, B, C and E) showed similar results,

211 with the germination rate varying between 64 and 69% (Table 1). For the following experiments,

212 we used stratification for 8 weeks at 3 °C, since this method was both fast and efficient

213 compared to the other methods tested. The exact moisture contents (MC) (both for the nutlet 214 and the seed inside) used in the experimentsDraft are shown in Table 2.

215 Tolerance to sub-zero storage

216 The tested temperatures (-3°, -18° and -196°C) had no significant effect on germination or

217 seedling emergence after one year of storage (Fig. 5). The best results after one year of storage

218 were obtained for nutlets kept at -3°C (68% and 69% for germination and seedling emergence,

219 respectively).

220 Optimal seeds hydration window for cryopreserved nutlets

221 Seeds dried in nutlets to 2.8% had a germination of 64% and a seedling emergence of 62%; in

222 comparison, in the control nutlets with a 10% MC, the germination was 71%, and the seedling

223 emergence was 64% (Fig. 3 and 4). Therefore no critical moisture content (CMC) was recorded.

224 The germination of the seeds with a nutlet MC of 2.8-18.1% submerged in LN varied from 58

225 to 70%, depending on the MC level, and did not differ significantly from the germination of the

226 seeds that were not treated with LN (63-76%; Fig. 2). Seedling emergence was between 56-70%

227 for the same range of nutlet MCs and did not differ significantly from seeds that were not treated

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228 with LN, which emerged at between 53 and 64% (Fig. 4). Nutlets with an MC above 18.1%

229 (20.8-29.6%) did not withstand freezing in LN and did not germinate; therefore, the HMFL was

230 established as 18.1% MC.

231 Discussion

232 Our study represents the first comprehensive assessment of the seed traits of Pterocarya

233 fraxinifolia, from its natural stands in Transcaucasia to assess the most appropriate ex situ

234 conservation method for this important and endangered relict tree. We focused on seeds,

235 examining their dormancy, storage behavior, desiccation tolerance and the possibility of

236 cryopreservation, which resulted in practical insights that are valuable for conservation

237 protocols in the future. Draft 238 Seed dormancy, desiccation and freezing tolerance

239 Our study confirms that seeds of the Caucasian wingnut are characterized by endogenous

240 nondeep physiological dormancy, which can be overcome by three weeks of cold stratification

241 at 4°C (Çiçek and Tilki 2008). However, to maximize seed germination, 5 to 8 weeks of cold

242 stratification is recommended, depending on the temperatures used during the germination

243 process. In comparison, seeds of P. stenoptera germinated at 16% without stratification, and

244 the highest germination success of 56% was recorded after just three weeks at 3 °C. The

245 prolongation of the cold period of stratification resulted in a decrease in germination (Grbić et

246 al. 2011). In contrast, Wang et al. (2018) reported 89% germination after four weeks of cold

247 stratification for the seeds of P. stenoptera. Our study confirmed that the 12 weeks of

248 stratification suggested by Young and Young (1992) can be shortened without a decrease in

249 germination. The use of LN in pretreatment as a low-temperature factor promoting the

250 permeability of the seed coat (Jastrzębowski et al. 2017) did not result in a significant increase

251 in germination. In stratification without a medium, we suggest not to over-moisturizing nutlets

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252 (above a 40% MC) because it could result in seed decay (Table 1) and therefore lower

253 germination. Our results indicate that in nursery practice, sowing nutlets after 8 weeks of cold

254 stratification with or without medium would be most effective.

255 According to our results, the dormant seeds of P. fraxinifolia maintained a high

256 germination and seedling emergence even after strong desiccation of the nutlet to 2.8% of MC;

257 therefore, we did not record a critical moisture content (CMC) for this species. Moreover, the

258 nutlets stored at subzero temperatures (-3, -18 and -196 °C) remained viable after one year of

259 storage. On the basis of these results, seeds of P. fraxinifolia can be classified as orthodox seeds.

260 The storage behavior of P. fraxinifolia seeds has never before been clearly determined in the

261 literature. In the Seed Information Database (Royal Botanic Gardens Kew 2018), seeds of P.

262 fraxinifolia are considered ‘likely orthodox’, but some authors (Young and Young 1992) refer

263 to safe storage for only for short periodsDraft of time without stating the proper moisture content or

264 a storage temperature. In some publications, seeds of P. fraxinifolia are mistakenly classified

265 as recalcitrant (Gordon and Rowe 1982; Grbić et al. 2011). These statements are based on only

266 brief observations and need clarification for the proper conservation of these species. In our

267 research, seeds of P. fraxinifolia were successfully stored at temperatures of -3, -18 and -196 °C

268 for one year at an MC level of 11.9%, which suggests the possibility of long-term storage of

269 the seeds in gene banks, both at traditionally used temperatures (-18 or -20 °C) and through

270 cryopreservation. Seed viability after longer storage times still needs to be examined in the

271 future.

272 Our work seems to confirm the results obtained for some other Juglandaceae family

273 members. The majority of these trees appears to produce orthodox or intermediate seeds,

274 although in the literature, there is often no unambiguous evidence. Gordon and Rowe (1982)

275 recommended storing seeds of Juglandaceae spp. at approximately 15% of their MC and above

276 0 °C, similar to many intermediate seed species. McIlwrick et al. (2000) described Juglans

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277 cincerea seeds as recalcitrant, which do not survive desiccation below a 15% MC or storage

278 below -40 °C. In general, seed behavior can differ among species in a specific family or even

279 in a genus; for example, in the genus Acer, most but not all of its members produce orthodox

280 seeds (Suszka et al. 1996). Sometimes differences in the seed desiccation tolerance for a given

281 species could be both geographical and taxonomical, as in Araucaria spp. (Tompsett 1983).

282 One of the confirmed seeds for ex situ storage of the genus Pterocarya was P. macroptera, in

283 which 89% of the viable seeds were found in seeds stored at 15% RH (ca. 5% seed MC) at -20

284 °C for one month (Royal Botanic Gardens Kew 2018). In our study, we examined the viability

285 of P. fraxinifolia seeds after one year at three different temperatures (including LN). Clear

286 descriptions of storage behavior are crucial for conservation programs. Occasionally, data

287 described in the literature can differ, as in the case of Carya illinoinensis, which was previously

288 thought to be a recalcitrant species (KingDraft and Roberts 1979) but was proven to be an orthodox

289 species, surviving desiccation below 5.3% (Bonner 1976; Dimalla and van Staden 1978; Goff

290 et al. 1992).

291 Feasibility of seed cryopreservation and nutlets storage

292 We examined whether P. fraxinifolia seeds could be effectively cryopreserved when the nutlets

293 were desiccated to an MC in a safe range of 2.8-18.1% before storage in LN. A similar range

294 of MC has been reported for some other orthodox species: Alnus glutinosa (2.7-19.2%), Ulmus

295 glabra (3.3-19.2%) and Betula pendula (2.0-23.2%) (Chmielarz 2010a, b, c). However, some

296 other riparian species, such as Salix caprea (8.5-23.4%; Popova et al. 2012) and Populus nigra

297 (10-15%, Michalak et al. 2015), have lower desiccation tolerances. The HMFL for those species

298 is similar to our results for the Caucasian wingnut (18.1%; Fig. 4). We described for the first

299 time the successful cryopreservation of seeds from the Juglandaceae family, although there

300 were many reports of the effective cryopreservation of the embryo axes from Juglans spp. and

301 Carya spp. (Pence 1990). Excised embryos of J. nigra can be cryopreserved up to two decades

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302 without a significant loss of the initial viability (Ballesteros and Pence 2019). Seedling

303 emergence in P. fraxinifolia did not differ when compared to germination, as occurred for

304 Hippophaë rhamnoides seeds (M. Wawrzyniak, personal communication), where seeds at some

305 MC levels after exposure to LN germinated but failed to establish healthy seedlings.

306 Although there is still a lack of information about the long-term storage of Pterocarya

307 and the majority of other Juglandaceae members, our research shows that no damage occurs

308 after one year of storage in LN (Fig. 1C, D). Thus, we assume that longer storage of P.

309 fraxinifolia is possible. Temperatures of - 3 and -18 °C, which are usually suggested for short-

310 and long-term storage in gene bank practice, also seem to be effective for P. fraxinifolia.

311 Cryopreservation offers the most stable conditions for seed storage, potentially for hundreds of

312 years (Pritchard et al. 2014). Draft 313 Conclusions

314 The seeds produced by P. fraxinifolia are classified as orthodox because they withstand

315 desiccation below 5% and can be stored at subzero temperatures for at least one year without

316 losing viability. Based on our results, seeds of P. fraxinifolia collected from natural endangered

317 stands can be germinated after 8 weeks of cold stratification, resulting in seedlings 6-7 weeks

318 after sowing. The safe range of MC allows for the effective storage of seeds in LN. Thus,

319 cryopreservation can be implemented in conservation programs and protocols for this highly

320 valuable relict tree species.

321 Acknowledgments

322 This work was supported by the Fondation Franklinia and by the Institute of Dendrology,

323 Polish Academy of Sciences, Kórnik, Poland (under statutory activity) (A.K.J, M.W., P.C.)..

324 We would like to thank two anonymous reviewers for valuable their comments, which greatly

325 improved this manuscript.

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Table 1 Germination (%)percentage of empty nutlets- and percentage of viable but non- germinated seeds after different stratification methods in 3 °C: A - 12 weeks; B - 8 weeks at;

C - 12 weeks without a stratification medium, with periodical soaking of seeds; D - 12 weeks without a stratification medium, without soaking; E - submergence in LN and stratification for

12 weeks (means with standard errors, SE).

Method Germination Empty nutlets Viable but non-

(%±SE) (%±SE) germinated seeds

(%±SE)

A 66.0±3.83 a 29.0±4.43 5.0±1.91

B 69.0±5.00 a 27.0±4.12 4.0±1.63

C 64.0±2.16 a 28.0±2.83 8.0±0.82

D 40.5±2.99 b Draft22.5±2.22 37.0±1.29

E 70.5±2.06 a 25.0±1.73 4.5±1.26

Different letters denote significant differences between treatments (P = 0.05, Tukey’s test).

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Table 2 Moisture content (MC) of the nutlets and seeds used in the experiment. MC in

stratification experiment was tested for methods without medium (see Table 1). Ten different

moisture levels (ranging from 3 to 30%) was used for establishing safe range of MC for

cryopreservation (means with standard errors, SE).

Moisture content (%±SE) Experiment Nutlets Seeds

After harvest MC 11.8±0.05 6.8±0.74

Stratification MC method D 52.3±2.27 26.6±1.96

method C 39.5±0.50 22.4±1.08

Cryopreservation 1 Draft2.8±0.03 2.3±0.06

2 6.4±0.02 4.1±0.11

3 9.2±0.12 5.0±0.14

4 11.9±0.03 5.8±0.07

5 15.2±0.11 7.8±0.18

6 18.1±0.20 9.8±0.18

7 20.8±0.12 11.6±0.14

8 24.3±0.03 13.6±0.21

9 27.6±0.40 15.7±0.37

10 29.6±0.24 17.4±0.31

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Figure list: Figure 1 (A) Caucasian wingnut fruit (a), nutlet (b) and a longitudinal section of a nutlet with a seed inside (c); (B) a seedling emerging from a nutlet; (C) seedling; (D) one-year-old seedling planted from a cryopreserved seed.

Figure 2 Germination rate of seeds after stratification: A – 8 weeks at 3 °C; B – 12 weeks at

3 °C; C – 12 weeks at 3 °C without stratification medium and 40% seed moisture content; D –

12 weeks at 3 °C without stratification medium and periodically submerged in water; E – submerged in liquid nitrogen (LN) for 48 h and stratified for 12 weeks at 3 °C.

Figure 3 Germination of Pterocarya fraxinifolia seeds after drying or moisturizing the nutlets to 10 levels of MC (2.8-29.6%), untreated (control) and treated (LN treated) for 48 h with liquid nitrogen (means from three replicationDraft with standard error bars); * - orthodox seeds; different letters denote significant differences between treatments (P = 0.05, Tukey’s test).

Figure 4 Emergence of Pterocarya fraxinifolia seedlings after drying or moisturizing the nutlets to 10 levels of MC (2.8-29.6%), untreated (conrtol) and treated (LN treated) for 48 h with liquid nitrogen (means from three replication with standard error bars); different letters denote significant differences between treatments (P = 0.05, Tukey’s test).

Figure 5 Seed germination and seedling emergence of Pterocarya fraxinifolia for nonstored seeds and seeds stored for two weeks and for one year at temperatures of -3, -18 and -196 °C;

A – germination; B – seedling emergence (means from three replication with standard error bars); different letters denote significant differences between treatments (P = 0.05, Tukey’s test).

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